Chronic liver disease and its common sequela cirrhosis, are growing public health concerns, and major risk factors for the development of hepatocellular cancer (HCC). HCC incidence and mortality is growing in the USA. Optimal medical therapies for HCC are lacking. FDA approved agents are modestly effective. Immune checkpoint inhibitors (ICIs) including PD-1 inhibitors Nivolumab and Pembrozilumab, have been both approved and show good response rates but only in a subset of HCC cases. Biomarkers of response remain unknown. Recent years have seen a revolution in HCC GWAS studies to identify molecular drivers. Mutations in CTNNB1 activate b-catenin in the Wnt pathway & seen in up to 37% of HCCs. However, b-catenin activation alone does not lead to HCC. Analysis of 2 large HCC cohorts (TCGA & French) revealed CTNNB1 mutations to co-occur with alterations in MET, MYC, TERT, NFE2L2, MLL2, ARID2 & APOB. Overexpression/activation of Met along with CTNNB1 mutations is seen in ~11% of HCCs. Coexpression of these genes in a subset of hepatocytes using sleeping beauty transposon/transposase (SB) & hydrodynamic tail vein injection (HTVI) led to HCC by 6 weeks in mice (hMet-b-catenin model). Gene expression analysis confirmed 70% similarity between hMet-b-catenin model and HCC patient subset with Met activation & CTNNB1 mutations. In aim 1, we will generate and characterize mouse models using SB/Crispr and HTVI to co-express mutant CTNNB1 and other genes frequently co-altered in subsets of human HCC including MYC, TERT, NFE2L2, MLL2, ARID2, & APOB. We already show HCC development in Met-b-catenin, MYC-b-catenin, TERT-b-catenin & NFE2L2-b- catenin, while others are ongoing. Comparison of gene expression between mouse models and human HCC subsets will validate the relevance of these models justifying a more comprehensive cellular & molecular characterization for innovative therapies. We will also test dependence of all mutant CTNNB1-mouse models to b-catenin by using of lipid nanoparticles (LNP) containing CTNNB1-siRNA (CTNNB1-LNP) to suppress b- catenin and assess response as we have shown for Kras-b-catenin (akin to hMet-b-catenin) model. In aim 2, we will focus on b-catenin-glutamine synthetase (GS)-glutamine-mTORC1 axis in mutant-CTNNB1 HCC, recently discovered and reported by us (Publication in Cell Metabolism). All mutant-CTNNB1 driven HCC models with all co-occurrences will be tested for response to mTORC1 inhibitors like Everolimus and RM-006 (novel exclusive mTORC1 inhibitor) and to upstream GS via genetic deletion of GS in established HCCs or via use of irreversible GS inhibitor L-methionine sulfoximine (MSO) and glutaminase inhibitor CB-839 that hampers production of glutamate, a substrate for GS to generate glutamine. In aim 3, we will investigate how to make b- catenin-active HCCs shown by us to be resistant to ICIs (publication in Cancer Discovery), sensitive to ICIs through use of combination therapy. Using an immunogenic Myc-lucOS-mutant-b-catenin HCC model, that is resistant to PD-1 inhibitor, we will test role of concomitant b-catenin suppression via CTNNB1-LNP, mTORC1 inhibition via Everolimus or GS inhibition via MSO, as sensitizing agents to PD-1 inhibitor. Thus overall, our proposal will develop clinically relevant and validated preclinical models utilizing mutant b-catenin as one cooperating oncogene and demonstrate role of mutant b-catenin in regulating tumor metabolism and immune microenvironment. Completion of ours study will thus provide justification for targeting b-catenin in a notable subset of HCC patients through innovative therapies and eventually pave the way for personalized medicine.